Arginine deiminase encapsulated inside erythrocytes and their use in treating cancer and arginase-1 deficiency

ABSTRACT

The present invention is related to arginine deiminase encapsulated into erythrocytes for use in therapy. It is in particular related to the use thereof in treating arginase-1 deficiency. Also, it relates to novel pharmaceutical compositions comprising arginine deiminase from  M. arginini  encapsulated into erythrocytes and the use thereof in treating diseases that may benefit from arginine depletion, such as arginine dependent cancers, in particular arginine-auxotrophic cancers, and arginase-1 deficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application is a divisional application of Ser. No.16/643,431, having a filing date of Feb. 28, 2020, which was a NationalStage application of International application PCT/EP2018/067679 filedJun. 29, 2018, which claims priority to the earlier filed Japanesepatent application No. 2017-254383 filed Dec. 28, 2017 and to Europeanpatent application No. 17306122.7 filed Aug. 31, 2017, all of saidapplications incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is related to arginine deiminase encapsulated intoerythrocytes for use in therapy. It is in particular related to the usethereof in treating arginase-1 deficiency. Also, it relates to novelpharmaceutical compositions comprising arginine deiminase from M.arginini encapsulated into erythrocytes and the use thereof in treatingdiseases that may beneficiate from arginine depletion, such as argininedependent cancers, in particular arginine-auxotrophic cancers, andarginase-1 deficiency.

BACKGROUND OF THE INVENTION

Arginine is a semi-essential amino acid. It is synthesized in the courseof the urea cycle from citrulline in two stages owing to the action ofarginosuccinate synthetase and arginosuccinate lyase. Arginine ismetabolized to ornithine under the action of arginase, and ornithine canin its turn be transformed into citrulline by a reaction catalyzed byornithine transcarbamylase.

It has been shown that certain types of tumor cells require arginine tobe supplied, and this led to consideration of arginine depletion being apossible treatment for these forms of cancers, calledarginine-auxotrophic cancers. Arginine deiminase, an arginine-degradingenzyme, catalyzes the hydrolyzation of arginine to citrulline andammonium by deamination of guanidine group. The antitumor activity ofarginine deiminase has been the subject of numerous publications. Thus,in vivo activity of arginine deiminase has been demonstrated withrespect to malignant melanoma and hepatocarcinoma. However, the enzymearginine deiminase has some major drawbacks.

Arginine deiminase is not produced in mammals but is obtained frommicroorganisms, making it a highly antigenic compound for mammals.

Moreover, this enzyme has a very short half-life in mammals, of theorder of about 5 hours, and must be administered daily at a high dose tobecome effective.

To overcome these drawbacks for the treatment of cancer European patentEP1874341 proposes an original approach which consists in encapsulatingarginine deiminase inside erythrocytes. EP1874341 notably discloses inexample 3 page 10 the entrapment of arginine deiminase originated fromPseudomonas aeruginosa.

Arginase-1 deficiency is a rare genetic disorder affecting the finalstep of the urea cycle in the liver that converts waste nitrogen in theform of ammonia into urea for excretion in the urine. It is caused bymutations in the ARG1 gene resulting in partial or complete loss ofarginase 1 enzyme which catalyzes the hydrolysis of arginine toornithine and urea (see FIG. 1).

Arginase-1 deficient patients exhibit hyperargininemia, progressiveneurological and intellectual impairment, persistent growth retardationand infrequent episodes of hyperammonemia.

There are currently no cures for arginase-1 deficiency and treatmentoutcomes are usually poor with a low-protein diet and/or nitrogenscavenger drugs. The main biochemical feature is accumulation ofarginine leading to toxic levels of guanidino compounds and nitric oxide(Sin et al, PLoS ONE 8(11)). Because arginine and its metabolites aresuspected to cause the neurologic phenotype in arginase-1 deficiency,the consensus goal of treatment has been reduction of plasma argininewith a low-protein diet, amino acid supplementation and administrationof nitrogen-scavenging agents. However, with this treatment strategy,normalization or near-normalization of plasma arginine in arginase-1deficiency is challenging.

Normalization of plasma arginine levels in arginase-1 deficiency istherefore challenging and the use of arginase enzyme therapy has beenpreviously investigated. Burrage et al., for instance, have investigateda modified PEGylated human recombinant arginase enzyme inarginase-deficient mouse models (Burrage et al. Human MolecularGenetics, 2015, 24(22)). Although this enzyme led to a reduction ofplasma arginine level it did not improve the survival ofarginase-deficient mice likely because of persistence of hyperammonemia.Hyperammonemia is the main treatment challenge in arginase deficiency(Burrage et al. Human Molecular Genetics 24(22)).

Therefore, the discovery of alternative ways for the depletion of plasmaarginine level in arginase deficiency has been a major goal for manyyears.

The reaction catalyzed by arginine deiminase is:

L-arginine+H₂O⇄L-citrulline+NH₃.

It is an equilibrated reaction and scientific arguments exist in thescientific community suggesting that using arginine deiminase to treatarginase-1 deficiency would not be possible, because:

-   -   This enzyme is of bacterial origin and may induce allergic        reactions    -   The products of the reaction catalyzed by arginine deiminase are        citrulline and ammoniac:    -   Citrulline can be converted back to arginine (see urea cycle in        FIG. 1), and    -   Ammoniac can be problematic in the context of arginase-1        deficiency where patients have episodes of hyperammonemia.

Contrary to those technical prejudices, the Applicant of the presentinvention unexpectedly found and demonstrated that arginine deiminaseencapsulated into erythrocytes may be a novel treatment approach toreduce the toxic accumulation of arginine and its metabolic sideproducts. The Applicant found, in particular, that the depletion ofserum arginine levels achieved in mice by administering the compositionof the present invention is high and therefore of therapeutic interest.The inventor demonstrated furthermore that said depletion of serumArginine levels in Arginine-1 deficient mice is not accompanied bynotable changes in ammonia levels. These results are especiallysurprising because of the existing technical prejudice according towhich an arginine deiminase would not enable the treatment of arginase-1deficiency because of the NH₃ production and a possible citrullineback-conversion.

Among the different arginine deiminase that can be encapsulated inerythrocytes, the applicant has identified a specific arginine deiminasethat has characteristics that lead to an unexpected improvement of PK/PDparameters.

In particular, arginine deiminase obtained from the bacteria Mycoplasmaarginini improves the PK/PD parameters. Moreover, said argininedeiminase from Mycoplasma arginini does not require any co-factor whichis a desirable characteristic for its activity inside the erythrocyte.The arginine deiminase from Mycoplasma arginini therefore has technicaladvantages over known arginine deiminase and is therefore very suitedfor the use in context of the present invention. In addition, thearginine deiminase from Mycoplasma arginini is a homodimer with amolecular weight of 92 kDa and thus favorable for entrapment insideerythrocytes.

Using said arginine deiminase will thus be a great advance in Argininedepletion therapies such as cancer therapy, in particulararginine-auxotrophic cancers, and arginase-1 deficiency therapy.

SUMMARY OF THE INVENTION

An object of the invention is thus a pharmaceutical compositioncomprising arginine deiminase encapsulated into erythrocytes, for itsuse in treating arginase-1 deficiency.

The invention refers, in particular, to a pharmaceutical compositioncomprising arginine deiminase encapsulated into erythrocytes and apharmaceutically acceptable vehicle, for its use in treating arginase-1deficiency.

In one preferred embodiment, the arginine deiminase encapsulated intoerythrocytes is from M. arginini.

Another object of the invention is a suspension of erythrocytesencapsulating arginine deiminase from M. arginini. Said suspension is inparticular a suspension of erythrocytes encapsulating arginine deiminasefrom M. arginini in a pharmaceutical acceptable vehicle.

A further object of the invention is a pharmaceutical compositioncomprising the suspension of the invention for its use in treatingarginase-1 deficiency or arginine-dependent cancers, treating orpreventing of septic shock, inhibiting angiogenesis and treatingangiogenesis associated diseases, in particular for its use in treatingarginase-1 deficiency or arginine-dependent cancers.

Another object of the invention is a method for treating arginase-1deficiency, comprising administering to a patient in need thereof aneffective amount of the pharmaceutical composition of the invention oradministering to a patient in need thereof an effective amount ofarginine deiminase encapsulated into erythrocytes.

Another object of the invention is a method for treating arginase-1deficiency or arginine-dependent cancers, treating or preventing ofseptic shock, inhibiting angiogenesis or treating angiogenesisassociated diseases comprising administering to a patient in needthereof an effective amount of the pharmaceutical composition comprisingarginine deiminase from M. arginini or an effective amount of argininedeiminase from M. arginini encapsulated into erythrocytes.

DETAILED DESCRIPTION Arginine Deiminase

“Arginine deiminase” also referred to as “ADI” is an enzyme thatcatalyzes the chemical reaction: L-arginine+H₂O ⇄L-citrulline+NH₃.Accordingly, in context of the present invention the arginine deiminaseused in context of the invention may also be referred to as “the enzyme”or ADI.

The two substrates of this enzyme are L-arginine and H₂O, whereas itstwo products are L-citrulline and NH₃. The arginine deiminase belongs tothe family of hydrolases and is identified under reference EC 3.5.3.6 inIUBMB Enzyme Nomenclature. ADI may originate from differentmicroorganisms, such as Bacillus pyocyaneus, Pseudomonas putida,Halobacterium salinarium, Mycoplasma arginini, Mycoplasma hominis,Pseudomonas aeruginosa, Lactobacillus lactis ssp. Lactis, andPseudomonas plecoglossicida (see Rui-Zhi Han et al., Appl. Microbiol.Biotechnol. 2016, 100: 4747-4760).

In one embodiment, the arginine deiminase is a protein.

As used herein, the term “protein” includes single-chain polypeptidemolecules as well as multiple-polypeptide complexes where individualconstituent polypeptides are linked by covalent or non-covalent means.

As used herein, the terms “polypeptide” and “peptide” refer to a polymerin which the monomers are amino acids and are joined together throughpeptide or disulfide bonds. The terms subunit and domain may also referto polypeptides and peptides having biological function.

The arginine deiminase employed in context of the present invention canbe of natural, synthetic or artificial origin, or obtained by geneticengineering (for example production of the enzyme in a host cell, forexample E. coli, after integration of a vector expressing the genecoding for the enzyme), and the person skilled in the art could refer,for example, to S. Misawa et al., J. of Biotechno. 36, 1994, 145-155 forthe description of production of ADI from M. arginini in E. coli.Arginine deiminases that can be used are described, for instance, inEP-A-1 011 717, EP-A-0 414 007, U.S. Pat. No. 5,372,942, JP-A-6062867,JP-A-2053490, JP-A-2035081. In an equivalent manner, the inventionincludes the use of analogues of this enzyme, such as variants andfragments, which can notably be enzymes that have been modified in orderto increase their enzymatic activity, as described for example in EP-A-0981 607.

The present invention employs preferably improved ADI for use in anydisease that benefits from the depletion of plasma arginine including,for example, arginine-dependent (auxotrophic) cancers and arginasedeficiency.

Accordingly, in one preferred embodiment, the arginine deiminase used incontext of the present invention is from Mycoplasma arginini. Thearginine deiminase from Mycoplasma arginini is a homodimer with amolecular weight of 92 kDa. The relative small size of said argininedeiminase in comparison to other arginine deiminases is advantageous forthe encapsulation in erythrocytes.

In one embodiment, the arginine deiminase from Mycoplasma argininicomprises the amino acid sequence of SEQ ID NO: 1 or variants orfragments thereof. The amino acid sequence of SEQ ID NO: 1 correspondsto the amino acid sequence of the arginine deiminase from Mycoplasmaarginini as available from the GenBank database under NCBI referencenumber WP_004416214.1, as available on Aug. 22, 2017.

References herein to amino acid sequences (also referred to aspolypeptides) include both, the particular amino acid sequences, andvariants of said sequences.

“Variant proteins” may be naturally occurring variants, such as splicevariants, alleles and isoforms, or they may be produced by recombinantmeans. Variations in amino acid sequence may be introduced bysubstitution, deletion or insertion of one or more codons into thenucleic acid sequence encoding the protein that results in a change inthe amino acid sequence of the protein. Optionally the variation is bysubstitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acids with any other amino acid in theprotein. Additionally or alternatively, the variation may be by additionor deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acids within the protein.

“Fragments” of the proteins are also encompassed by the invention. Suchfragments may be truncated at the N-terminus or C-terminus, or may lackinternal residues, for example, when compared with a full lengthprotein. Certain fragments lack amino acid residues that are notessential for enzymatic activity. Preferably, said fragments are atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 250,300, 350, 380, 400, or more amino acids in length.

Variant proteins may include proteins that have at least about 80% aminoacid sequence identity with a polypeptide sequence disclosed herein.Preferably, a variant protein will have at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% amino acid sequence identity to a polypeptide sequence asdisclosed herein. Amino acid sequence identity is defined as thepercentage of amino acid residues in the variant sequence that areidentical with the amino acid residues in the reference sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Sequenceidentity may be determined over the full length of both variant andreference amino acid sequences.

It will be understood by a skilled person that numerous differentnucleic acids can encode the same polypeptide as a result of thedegeneracy of the genetic code. In addition, it is to be understood thatskilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the polypeptide sequence encoded by thenucleic acids of the invention to reflect the codon usage of anyparticular host organism in which the polypeptides are to be expressed.Nucleic acids encoding the ADI used in context of the invention may bemodified by any method available in the art. Nucleic acids encoding theADI used in context of the invention may be produced recombinantly,synthetically, or by any means available to those of skill in the art.They may also be cloned by standard techniques such as PCR (polymerasechain reaction).

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid.

In the context of the present application, the percentage of identity iscalculated using a global alignment (i.e. the two sequences are comparedover their entire length). Methods for comparing the identity of two ormore sequences are well known in the art. The “needle” program, whichuses the Needleman-Wunsch global alignment algorithm (Needleman andWunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment(including gaps) of two sequences when considering their entire length,may for example be used. The needle program is for example available onthe ebi.ac.uk world wide web site. The percentage of identity inaccordance with the invention is preferably calculated using theEMBOSS::needle (global) program with a “Gap Open” parameter equal to10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical” to a reference sequence maycomprise mutations such as deletions, insertions and/or substitutionscompared to the reference sequence. In case of deletions, insertionsand/or substitutions, the protein consisting of an amino acid sequenceat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to areference sequence may correspond to a homologous sequence derived fromanother species than the reference sequence.

Amino acid substitutions may be conservative or non-conservative.Preferably, substitutions are conservative substitutions, in which oneamino acid is substituted for another amino acid with similar structuraland/or chemical properties. The substitution preferably corresponds to aconservative substitution as indicated in table 1 herein below.

TABLE 1 Conservative amino acid substitution Conservative substitutionsType of Amino Acid Ala, Val, Leu, Ile, Amino acids with aliphatic Met,Pro, Phe, Trp hydrophobic side chains Ser, Tyr, Asn, Gln, Cys Aminoacids with uncharged but polar side chains Asp, Glu Amino acids withacidic side chains Lys, Arg, His Amino acids with basic side chains GlyNeutral side chain

Preferably, said variants or fragments retain a biological activity of aprotein having the full-length amino acid sequence of SEQ ID NO 1.

In some embodiments, the arginine deiminase used in context of theinvention might be a variant or fragment having an increased biologicalactivity or enzymatic activity in comparison to a protein having thefull-length amino acid sequence of SEQ ID NO 1. Accordingly, saidvariant or fragment might comprise amino acid substitutions that improveits enzymatic activity. “Improved enzymatic activity” herein refers, forexample, to the improvement of the pH optimum, improvement of the Km andkcat values and an improved thermostability. Such mutations are eitherknown to the skilled in the art or can be easily derived by the skilledin the art from the general knowledge as disclosed, for example, inRui-Zhi Han et al. (Appl. Microbiol. Biotechnol. 2016, 100: 4747-4760).

In one embodiment, the arginine deiminase variant or fragment thereofmight be modified in order to increase the plasma half-life in vivo.

Pharmaceutical Compositions or Suspensions

The terms “Pharmaceutical composition” or “therapeutic composition” asused herein refer to a compound or composition capable of inducing adesired therapeutic effect when properly administered to a patient.

The invention refers to a pharmaceutical composition and a suspension,both comprising arginine deiminase encapsulated in erythrocytes. Thedefinitions given in the present description refer to the compositionsand the suspensions of the invention. The erythrocytes encapsulating ADIare preferably in suspension in a pharmaceutically acceptable vehicle.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate.

According to a particular embodiment, the “pharmaceutically acceptablevehicle” is a “preservation solution” or a “preservative solution forerythrocytes”, i.e. a solution in which the erythrocytes encapsulatingthe enzyme are suspended in their suitable form for being stored whileawaiting their injection. A preservation solution preferably comprisesat least one agent promoting preservation of the erythrocytes, notablyselected from glucose, dextrose, adenine and mannitol.

In one embodiment, the preservation solution is an aqueous solutioncomprising NaCl and/or adenine. In a further embodiment, thepreservation solution further comprises at least one compound selectedfrom the group consisting of glucose, dextrose and mannitol. As anexample, it may comprise NaCl, adenine and dextrose. In one example, thepreservative solution is an AS3 medium. As another example, it maycomprise NaCl, adenine, glucose and mannitol. In a further example, thepreservative solution is a SAGM medium, such as SAG-Mannitol, or a ADsolmedium.

The exact form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and gender of the patient, etc. It will be further understood that thecomposition and suspensions of the invention are intended forintravenous or intra-arterial administration, preferably for injection,infusion or perfusion. Accordingly, the pharmaceutical composition ofthe invention is formulated for intravenous or intra-arterialadministration.

The compositions of the invention can be ready for use or not. Theready-for-use pharmaceutical composition of the invention has ahematocrit suitable for administration without dilution. When not readyfor use, the composition can also be packaged with a higher hematocritvalue such that it has to be diluted before administration.

The “hematocrit (Ht or HCT)” is generally known as the volume percentage(vol %) of red blood cells in blood. In context of the presentinvention, the hematocrit may further refer to the volume percentage(vol %) of red blood cells in the composition or suspension referred to,such as the pharmaceutical composition or the suspension of theerythrocytes.

According to the invention, the hematocrit of the ready-for-usepharmaceutical composition advantageously lies between 40 and 70%,preferably between 45 and 65%, more preferably between 45 and 55%, suchas 46 and 54%, 47 and 53%, 48 and 52%, for example about 50%. When notready-for-use the hematocrit of the to-be-diluted pharmaceuticalcomposition can be higher, in particular lying between 50 and 90%,preferably 60 and 90%. In a connected embodiment, the dilution of thepharmaceutical composition to obtain the hematocrit values of theready-to-use composition, defined herein above, may be made with thepharmaceutically acceptable vehicle.

“Erythrocytes” are also referred to as red blood cells. The erythrocytesdevelop in the bone marrow and circulate for about 100-120 days in thebody before their components are recycled by macrophages. Nearly half ofthe blood's volume (40% to 45%) is red blood cells. Methods to isolateerythrocytes are known to the skilled in the art.

In one embodiment, the erythrocytes are issued from a mammal of the samespecies than the treated subject or patient. When the mammal is a human,the erythrocytes are preferably of human origin. In an embodiment, theerythrocytes come from the patient itself.

In another embodiment, stem cells are used to generate the erythrocytes.The person skilled may refer to Russeau et al. (ISBT Science Series(2016) 11 (Suppl. 1), 111-117). The stem cells may further betransformed to express the enzyme inside the erythrocytes. The personskilled in the art may further refer to US2015/0182588. These documentsare incorporated herein by reference.

The arginine deiminase used in context of the invention is highlyactive. Activity may be expressed using, for example, the unit U. The“unit (U)” refers to the amount of enzyme that converts 1 μmole ofsubstrate per minute. Accordingly, in context of the present invention1U refers to the amount of enzyme that converts 1 μmole of arginine perminute, preferably, at 37° C. More preferably, 1 U refers to theactivity of the arginine deiminase when present in the buffers usuallyused for measuring the activity of arginine deiminase, such as thebuffers specified in (Boyde and Rahmatullah, 1980, AnalyticalBiochemistry, vol 107, p 424-431). In some embodiments, the “unit” asdefined herein above may also be referred to as “international unit(IU)”. Accordingly, in some embodiments 1U and 11U are interchangeable.

Methods to qualitatively and/or quantitatively evaluate the activity ofan arginine deiminase are known to the skilled in the art. Typically,the activity of arginine deiminase is measured as described in detailherein below in the experimental section, in particular in example 1 and2 of the experimental section. The exact experimental conditions used tomeasure the activity of arginine deiminase are known to the skilled inthe art and for example described in (Boyde and Rahmatullah, 1980,Analytical Biochemistry, vol 107, p 424-431).In one embodiment, thearginine deiminase used in context of the invention has, prior toencapsulation, a specific activity of 10 to 100 U/mg, such as 20 to 80,30 to 70, 40 to 60 U/mg, wherein the mg refer to the amount of purifiedenzyme.

“Purified enzyme” herein refers to an enzyme having a purity of 90 to100%, such as 92 to 100%, 94 to 100%, more than 96%, more than 97%, morethan 98%, more than 99%, for example 97%. Methods to determine thepurity of an enzyme are known to the skilled in the art and include, forexample, SDS gel analysis, or Mass spectrometry analysis, preferably SDSgel analysis.

“Arginine deiminase is encapsulated into erythrocytes” means that thearginine deiminase is contained within the erythrocytes.

In one embodiment, the concentration of encapsulated arginine deiminasein context of the present invention, also referred to asintra-erythrocyte concentration of arginine deiminase, is from 0.1 to 7mg/ml, preferably 0.5 to 6.5 mg/ml, such as 1 to 6 mg/ml, 1 to 5 mg/ml,1 to 4 mg/ml, 1 to 3 mg/ml, more preferably, 1.2 to 2.8 mg/ml, 1.4 to2.6 mg/ml, 1.6 to 2.4 mg/ml.

In some embodiments, the concentration of encapsulated argininedeiminase is at least 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2,2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 6 mg/ml. In some embodiments, theconcentration of encapsulated arginine deiminase is at most 7, 6.5, 5,5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2 mg/ml.

In another embodiment, the concentration of encapsulated argininedeiminase is from 1 to 1000 U/ml, preferably 1 to 500 U/ml, such as 1 to400 U/ml, 5 to 400 U/ml, 5 to 350 U/ml.

In some embodiments, the concentration of encapsulated argininedeiminase is at least 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200to 400 U/ml. In some embodiments, the concentration of encapsulatedarginine deiminase is at most 1000, 800, 600, 500, 400, 350, 300, 200,100 U/ml.

Preferably, said concentration of encapsulated arginine deiminase, inmg/ml or in U/ml, refers to the intra-erythrocyte concentration ofarginine deiminase per ml of the pharmaceutical composition of theinvention or the suspension of the invention, more preferably, refers tothe intra-erythrocyte concentration of the erythrocytes per ml of theerythrocyte solution of the composition or suspension, wherein saidsolution has typically a hematocrit of 50%.

In one embodiment, the composition or suspension of the invention, in apreservation solution, is characterized by an extracellular hemoglobinlevel maintained at a level equal to or less than 0.5, in particular0.3, notably 0.2, preferably 0.15, even better 0.1 g/dl at 72 h andpreservation at a temperature comprised between 2 and 8° C.

In particular, the composition or suspension of the invention, in apreservation solution, is characterized by an extracellular hemoglobinlevel maintained at a level equal to or less than 0.5, in particular0.3, notably 0.2, preferably 0.15, even better 0.1 g/dl for a periodcomprised between 24 h and 20 days, notably between 24 and 72 h andpreservation at a temperature comprised between 2 and 8° C.

The “extracellular hemoglobin level” is advantageously measured by themanual reference method described in G. B. Blakney and A. J. Dinwoodie,Clin. Biochem. 8, 96-102, 1975. Automatic devices also exist whichallows this measurement to be made with a sensitivity which is specificto them.

In particular, the composition or suspension of the invention, in apreservation solution, is characterized by a hemolysis rate maintainedat equal to or less than 2, notably 1.5, preferably 1% at 72 h andpreservation at a temperature comprised between 2 and 8° C.

In particular, the composition or suspension, in a preservationsolution, is characterized by a hemolysis rate maintained at equal to orless than 2, notably 1.5, preferably 1% for a period comprised between24 h and 20 days, notably between 24 and 72 h and at a temperaturecomprised between 2 and 8° C.

In one embodiment, the composition of the invention is a suspension. Inone embodiment, the suspension of the invention and the suspension incontext of the invention have an osmolarity of between 270 and 350mOsm/1. Methods to measure osmolarity are known to the skilled in theart. In one example, the osmolarity is measured with an osmometer(Micro-Osmometer Loser Type 15). The osmolarity is preferably measuredat standard conditions (i.e. at 25° C. and 1 atm).

Therapeutic Methods and Uses

The inventors of the present invention have shown that a pharmaceuticalcomposition comprising arginine deiminase encapsulated into erythrocytesdecreases blood L-Arginine levels in arginase deficient mice by up to73% three days after administration, for instance, when administered ata dose of 8 ml/kg. It will be understood by the skilled in the art thatthese results demonstrate that the composition of the invention, as wellas the suspension of the invention may be used to treat arginase-1deficiency, because the elevated level of serum arginine is directlyrelated to clinical condition of the subject or patient which istreated.

The inventors further demonstrated in mice that surprisinglyadministering the composition or suspension of the invention does notnotably changes ammonia levels in the treated mice and thus potentiallydoes not affect eventual episodes of hyperammonemia in the subject orpatient that is treated.

Accordingly, the present invention refers to a pharmaceuticalcomposition comprising arginine deiminase encapsulated into erythrocytesand a pharmaceutically acceptable vehicle, for its use in treatingarginase-1 deficiency. The invention further refers to a suspension oferythrocytes encapsulating arginine deiminase, in particular argininedeiminase from M. arginini, for its use in treating arginase-1deficiency.

“Arginase-1 deficiency” is an inherited metabolic disease in which thebody is unable to process arginine (a building block of protein). Itbelongs to a group of disorders known as urea cycle disorders. Theseoccur when the body's process for removing ammonia is disrupted, whichcan cause ammonia levels in the blood to rise (hyperammonemia). In mostcases, symptoms appear between the ages of one and three years. Symptomsmay include feeding problems, vomiting, poor growth, seizures, and stiffmuscles with increased reflexes (spasticity). People with arginase-1deficiency may also have developmental delay, loss of developmentalmilestones, and intellectual disability. Arginase-1 deficiency is causedby mutations in the ARG1 gene and is inherited in an autosomal recessivemanner. Most people with arginase-1 deficiency appear to be healthy atbirth and have normal development during early childhood. The firstfeatures of arginase-1 deficiency often appear between the ages of oneand three years. In some cases, symptoms may begin earlier or later.

Arginase-1 deficiency may be characterized by one or more signs andsymptoms selected from the list including poor growth (present in allthe people who have arginase deficiency), stiff muscles and increasedreflexes (spasticity), developmental delay, loss of previously acquireddevelopmental milestones, intellectual disability, seizures, small headsize (microcephaly), problems with balance and coordination, behavioralabnormality, diaminoaciduria, Intellectual disability (severe),neurological speech impairment, EEG (Electroencephalogram) abnormality,hemiplegia/hemiparesis, hyperammonemia, progressive spastic quadriplegiaand seizures.

“Methods to diagnose Arginine 1 deficiency” are known to the skilled inthe art and usually include a full-panel expanded newborn screeningtesting. Typically, three- to fourfold elevation of plasma arginineconcentration above the upper limit of normal is highly suggestive ofthe diagnosis. The diagnosis is further typically confirmed byidentification of biallelic ARG1 pathogenic variants on moleculargenetic testing or failure to detect arginase enzyme activity (usually<1% of normal) in red blood cell extracts.

The invention further refers to a method for treating arginase-1deficiency comprising administering to a patient in need thereof aneffective amount of arginine deiminase encapsulated into erythrocytes oran effective amount of the composition of the invention.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition.

Accordingly, the term “treatment” not only includes treatment leading tocomplete cure of the disease, but also treatments slowing down theprogression of the disease and/or prolonging the survival of thepatient.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A therapeutically effective amount of the composition or suspension ofthe invention may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the protein,to elicit a desired therapeutic result. A therapeutically effectiveamount encompasses an amount in which any toxic or detrimental effectsof the inhibitor are outweighed by the therapeutically beneficialeffects. A therapeutically effective amount also encompasses an amountsufficient to confer benefit, e.g., clinical benefit.

In some embodiments, the methods of the invention comprise theadministration of the composition of the invention as defined hereinabove, in combination with at least one further treatment which isdirected against arginase-1 deficiency, either sequentially orsimultaneously.

One further treatment directed against arginase-1 deficiency hereinrefers to low-protein diet, administration of nitrogen scavengers,injections of botulinum toxin, or the administration of amino acids suchas ornithine or lysine.

“Nitrogen scavengers” include, but are not limited to, benzoate,phenylbutyrate, and phenylacetate.

As indicated herein above, the inventors of the present inventiondemonstrated that the arginine deiminase of M. arginini encapsulated inErythrocytes decreases blood L-arginine levels in arginase-1 deficientmice by up to 73% three days after administration, for instance whenadministered at a dose of 8 ml/kg.

Accordingly, it will be understood by the skilled in the art that apharmaceutical composition comprising a suspension of erythrocytesencapsulating arginine deiminase from M. arginini is not only efficientin the treatment of arginase-1 deficiency but also in the treatment ofother diseases in which lowering the concentration of blood argininemight be beneficial.

Accordingly, the invention finds particularly interesting application inarginine-dependent cancers, for which the favorable effect of plasmaarginine depletion has been demonstrated (see for example F. Izzo etal., 2004, C. M. Ensor et al., 2002, F. W. Holtsberg et al., 2002, J. S.Bomalaski et al., 2003, and Curley S. A. et al., 2003, previouslycited).

However, plasma arginine depletion may also be beneficial for treatingor preventing of septic shock, inhibition of angiogenesis and associateddiseases.

Accordingly, in one embodiment, the present invention refers to apharmaceutical composition comprising a suspension of erythrocytesencapsulating arginine deiminase from M. arginini for its use inlowering the concentration of blood arginine.

Accordingly, in a related embodiment, the present invention refers to apharmaceutical composition comprising arginine deiminase from M.arginini encapsulated into erythrocytes and a pharmaceuticallyacceptable vehicle for its use in lowering the concentration of bloodarginine

The definitions referring to the “pharmaceutical compositions”, the“arginine deiminase”, “suspension”, “pharmaceutically acceptablevehicle” are as given herein above and apply mutatis mutandis.

In particular, lowering the concentration of blood arginine is useful intreating arginase-1 deficiency, treating arginine-dependent cancers,treating or preventing a septic shock, for the inhibition ofangiogenesis and the treatment of associated diseases.

Accordingly, in one embodiment, the invention refers to a pharmaceuticalcomposition comprising a suspension of erythrocytes encapsulatingarginine deiminase from M. arginini for its use in treating arginase-1deficiency, treating arginine-dependent cancers, treating or preventingof septic shock, inhibition of angiogenesis and associated diseases.

Accordingly, in a related embodiment, the present invention refers to apharmaceutical composition comprising arginine deiminase from M.arginini encapsulated into erythrocytes and a pharmaceuticallyacceptable vehicle for its use in treating arginase-1 deficiency,treating arginine-dependent cancers, treating or preventing of septicshock, inhibition of angiogenesis and associated diseases.

“Arginase-1 deficiency” is as defined herein above.

“Arginine-dependent cancers” refer to cancers involving cancer cellsthat require arginine for replication, those tumors are unable tosynthesize some or all of the arginine that they need, mostly due to thelack of the biosynthetic enzyme asparagine synthetase and thereforerequire a supply of arginine, therefore those cancers are also referredto as “arginine auxotrophic cancers”. Plasma arginine depletion willdeprive these cells of the arginine that is essential for theirdevelopment, leading to targeted death of these cells, inhibition oftumor growth or regression of the tumor mass.

Arginine dependent cancers may be selected from the group consisting ofhepatocarcinoma, primary liver cancer, melanoma, breast cancer,neuroblastoma, leukemia, mesothelial cancer, urological cancer, sarcoma,gastric cancer and cerebral cancer.

“Mesothelial cancer” refers, for instance, to malignant pleuralmesothelioma (MPM).

“Urological cancer” includes, for instance, renal cell carcinoma,prostate cancer, colon cancer and bladder cancer, in particulartransitional cell carcinoma.

“Sarcoma” refers, for example, to Astrocytoma, Oligodendroglioma.

“Cerebral cancer” refers, for example, to osteosarcoma.

In one aspect, the arginine dependent cancer is hepatocarcinoma orprimary liver cancer. In a further aspect, the arginine dependent canceris melanoma, in particular, malignant melanoma, in its various forms,such as superficial spreading melanoma and nodular melanoma. Accordingto a further aspect, the arginine dependent cancer is one of thefollowing forms of cancer:

breast cancer,

neuroblastoma (Gong H. et al., Int. J. Cancer 2003, 106: 723-8),

leukemia (Gong H. et al. Leukemia 2000, Vol. 14, 826-9; Noh E. J. etal., Int. J. Cancer 2004, 112: 502-8).

“Treating” is as defined herein above.

By “preventing” or “Prevention” is meant a prophylactic use (i.e. on anindividual susceptible of developing a given disease).

“Inhibition of angiogenesis and the treatment of associated diseases”refers to the treatment of diseases such as: angioma, angiofibroma,arthritis, diabetic retinopathy, retinopathy of the premature,neovascular glaucoma, disease of the cornea, involutional and otherforms of macular degeneration, pterygium, retinal degeneration,retrolental fibroplasia, psoriasis, telangiectasia, granulomapyogenicum, seborrheic dermatitis, acne, cancer and metastases connectedwith angiogenesis (WO0209741; Park I. S. et al., Br. J. Cancer 2003, 89:907-14).

A further object of the invention is the use of erythrocytesencapsulating ADI or a suspension of such erythrocytes for thepreparation of a medicament intended for the treatment of the diseasespresented herein.

This use takes account of the characteristics presented for thesuspensions and the pharmaceutical compositions of the invention ormedicament.

Another object of the invention is a method for treating arginase-1deficiency, comprising administering to a patient in need thereof aneffective amount of the pharmaceutical composition of the invention oradministering to a patient in need thereof an effective amount ofarginine deiminase encapsulated into erythrocytes or administering to apatient in need thereof an effective amount of the medicament accordingto the invention.

Another object of the invention is a method for treating arginase-1deficiency or arginine-dependent cancers, treating or preventing ofseptic shock, inhibiting angiogenesis or treating angiogenesisassociated diseases comprising administering to a patient in needthereof an effective amount of the pharmaceutical composition comprisingarginine deiminase from M. arginini or an effective amount of argininedeiminase from M. arginini encapsulated into erythrocytes oradministering to a patient in need thereof an effective amount of themedicament according to the invention.

The method of treatment comprises the administration to a patient inneed thereof of a “therapeutically effective dose,” “therapeuticallyeffective amount,” or “effective amount”, wherein an effective amount isas defined herein above.

In particular, a “therapeutically effective dose,” “therapeuticallyeffective amount,” or “effective amount” can be routinely determined bythose of skilled in the art. The amount of the enzyme actuallyadministered may be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered theage, weight, and response of the individual patient, the severity of thepatient's symptoms, etc. The effective amounts may typically besufficient to induce depletion of arginine in the blood circulation.Preferably, this depletion may correspond to maintaining arginine belowa threshold level during a sufficient amount of time.

In some examples, the arginine level may be maintained below a thresholdlevel for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10 days, preferably for atleast 3 days.

Typical effective amounts of enzyme are further detailed herein below inthe section “dose”.

The pharmaceutical compositions can be administered by intravenous orintra-arterial injection and preferably by injection, infusion orperfusion from a blood bag or the like. Administration is typicallyaffected intravenously into the arm or via a central catheter.

In particular, from about 10 to about 250 ml of suspension (one dose),pharmaceutical composition or medicament according to the invention isadministered. Beyond 20 ml, use of infusion or perfusion is preferred.

A treatment comprises the administration of one dose or of several dosesaccording to the protocol decided. This can provide for severaladministrations at monthly, fortnightly or weekly intervals, preferablyonce, twice or three times a week, over the recommended duration of thetreatment.

In one embodiment, the treatment in context of the invention can consistin the administration of one dose, as defined herein below in thesection dose, each time (each dose).

In one example, the treatment in context of the invention can consist inthe administration of the equivalent of 0.001 mg/kg of enzyme per kg ofbody weight to 1000 mg/kg of enzyme per kg of body weight, preferably0.01 mg/kg of enzyme per kg of body weight to 500 mg/kg of enzyme per kgof body weight, preferably, each time (each dose). Preferably, from 0.01mg/kg of enzyme per kg of body weight to 200 mg/kg of enzyme per kg ofbody weight is administered, preferably, each time (each dose).

In a further example, as further specified herein below in the sectiondose, the treatment in context of the invention can consist in theadministration of the equivalent of 10 U/kg of patient body weight and100000 U/kg of enzyme per kg of body weight each time (each dose).Preferably, from 10 U/kg of patient body weight and 15000 U/kg of enzymeper kg of body weight each time (each dose). More preferably, from 500U/kg of patient body weight and 3500 U/kg of enzyme per kg of bodyweight each time (each dose).

Doses

The compositions of the invention are preferably packaged at orpresented in a volume containing a pre-determined quantity of activematerial calculated to produce the desired therapeutic effect. It willbe understood by the skilled in the art, that volume or dose of thecomposition to be administered depends, for example, on the condition ofthe mammal intended for administration (e. g., weight, age, sex andhealth, concurrent treatment, if any, frequency of treatment), the modeof administration and the type of formulation.

In context of the present invention, the volume in which the compositionfor administration is packaged is also referred to as a dose. In oneembodiment, said volume is from 10 to 250 ml.

Accordingly, the composition for administration is preferably packagedat or presented in one dose; preferably said dose has a volume asdefined herein above. The packaging is preferably in a container, suchas, for instance a blood bag of the type suitable for a bloodtransfusion and the like. The whole of the quantity of encapsulatedenzyme corresponding to the medical prescription is preferably containedin said container. In other words, one dose of erythrocytesencapsulating a given amount of enzyme may be present in one containeror pharmaceutical composition. In one embodiment, this one dose isintended to be fully administered to a patient in need thereof. Inanother embodiment, the administration may be achieved in a single doseor several doses.

The amount of enzyme encapsulated in one dose may be between 0.001 mg/kgof patient body weight and 1000 mg/kg of patient body weight. Morepreferably, the amount of enzyme encapsulated in one dose is between0.01 mg/kg of patient body weight and 1000 mg/kg of patient body weight.Most preferably, the amount of enzyme encapsulated in one dose isbetween 0.01 mg/kg of patient body weight and 500 mg/kg of patient bodyweight. Most preferably, the amount of enzyme encapsulated in one doseis between 0.01 mg/kg of patient body weight and 200 mg/kg of patientbody weight or between 0.01 mg/kg of patient body weight and 100 mg/kgof patient body weight.

In some embodiments, the amount of enzyme encapsulated in one dose is atleast 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50 mg/kg of patient body weight. In some embodiments, theamount of enzyme encapsulated in one dose is at most 200, 150, 100, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 25, 15, 10, 5, 1, 0.5, 0.1 mg/kg ofpatient body weight.

The amount of enzyme encapsulated in one dose may also be between 10U/kg of patient body weight and 100000 U/kg of patient body weight. Morepreferably, the amount of enzyme encapsulated in one dose is between 10U/kg of patient body weight and 80000 U/kg of patient body weight. Mostpreferably, the amount of enzyme encapsulated in one dose is between 10U/kg of patient body weight and 50000 U/kg of patient body weight. Mostpreferably, the amount of enzyme encapsulated in one dose is between 10U/kg of patient body weight and 5000 U/kg of patient body weight, 50U/kg of patient body weight and 3500 U/kg of patient body weight, orbetween 50 U/kg of patient body weight and 3500 U/kg of patient bodyweight.

In some embodiments, the amount of enzyme encapsulated in one dose mayalso be of at least 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000UI/kg of patient body weight. In some embodiments, the amount of enzymeencapsulated in one dose may also be of at most 10000, 9500, 9000, 8500,8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500,2000, 1500, 1000, 900, 800, 600, 500, 450, 400, 350, 300, 250, 200, 150,100 UI/kg of patient body weight.

It will be understood by the skilled in the art, that the doses used forthe administration can be adapted as a function of various parameters,and in particular as a function of the mode of administration used, ofthe relevant pathology, or alternatively of the desired duration oftreatment.

A pharmaceutical composition comprising arginine deiminase encapsulatedinto erythrocytes and a pharmaceutically acceptable vehicle has beendescribed in the prior art in context of the treatment of cancers.Adaptation of the dosages described in the above identified publicationsto the compositions of the invention for use in in treating arginase-1deficiency are within the capabilities of the person skilled in the art.

Methods of Encapsulation

The Erythrocytes may be obtained as described herein above in thesection “Pharmaceutical compositions”.

Encapsulating the enzymes into erythrocytes may be performed using anerythrocyte suspension that is put into contact with a hypotonic liquidmedium resulting in the opening of pores in the erythrocyte membrane.There exist three alternatives in the lysis-resealing technique, whichare hypotonic dialysis, hypotonic preswelling and hypotonic dilution,all based on the difference in osmotic pressure between the inside andthe outside of the erythrocytes. Hypotonic dialysis is preferred.

The suspension of erythrocytes encapsulating the enzyme is notably ableto be obtained with the following method:

1—suspending a pellet of erythrocytes in an isotonic solution at ahematocrit level equal to or greater than 65%, cooling between +1 and+8° C.,

2—a lysis procedure, at a temperature maintained between +1 and +8° C.,comprising the passing of the suspension of erythrocytes at a hematocritlevel equal or greater than 65% and of a cooled hypotonic lysis solutionbetween +1 and +8° C., into a dialysis device, such as a coil or adialysis cartridge (the cartridge is preferred);

3—an encapsulation procedure by adding, preferably gradually, the enzymeto be encapsulated (notably in a solution made up beforehand) into thesuspension before or during lysis, at a temperature maintained between+1 and +8° C.; and

4—a resealing procedure conducted in the presence of an isotonic orhypertonic, advantageously hypertonic solution, at a higher temperature,notably comprised between +30 and +42° C.

In a preferred alternative, use may be done of the method described inWO-A-2006/016247 (EP 1 773 452; which is incorporated herein byreference):

1—suspending a pellet of erythrocytes in an isotonic solution at ahematocrit level equal to or greater than 65%, cooling between +1 and+8° C.,

2—measuring osmotic fragility from a sample of erythrocytes from thissame pellet,

3—a lysis procedure, at a temperature maintained between +1 and +8° C.,comprising the passing of the suspension of erythrocytes at a hematocritlevel equal to or greater than 65% and of a hypotonic lysis solutioncooled between +1 and +8° C., into a dialysis device, such as a coil ora dialysis cartridge (the cartridge is preferred); the lysis parametersbeing adjusted according to the osmotic fragility measured earlier;notably, depending on the measured osmotic fragility, the flow of theerythrocyte suspension passing into the dialysis device is adjusted orthe osmolarity of the lysis solution is adjusted; and

4—a procedure for encapsulation by adding, preferably gradually, theenzyme to be encapsulated (notably in a solution made beforehand) in thesuspension before and during lysis, at a temperature maintained between+1 and +8° C.; and

5—a resealing procedure conducted in the presence of an isotonic orhypertonic, advantageously hypertonic solution, at a higher temperature,notably comprised between +30 and +42° C.

Notably, for dialysis, the pellet of erythrocytes is suspended in anisotonic solution with a high hematocrit level, equal to or greater than65%, and preferably equal to or greater than 70%, and this suspension iscooled between +1 and +8° C., preferably between +2 and +6° C.,typically around +4° C. According to a particular method, the hematocritlevel is comprised between 65 and 80%, preferably between 70 and 80%.

When it is measured, the osmotic fragility is advantageously measured onerythrocytes just before the lysis step, in the presence or in theabsence, preferably in the presence of the enzyme to be encapsulated.The erythrocytes or the suspension containing them are advantageously ata temperature close to, or identical with the temperature selected forlysis. According to another advantageous feature of the invention, theconducted measurement of the osmotic fragility is rapidly utilized, i.e.the lysis procedure is carried out in a short time after taking thesample. Preferably, this lapse of time between the sampling andbeginning of lysis is less than or equal to 30 minutes, still betterless than or equal to 25 and even to 20 minutes.

As regards performing the lysis-resealing procedure with measurement andaccounting for the osmotic fragility, one skilled in the art may referfor more details to WO-A-2006/016247. This document is incorporatedherein by reference in its entirety.

An improvement of this encapsulation technique was described in WO2014/180897, to which one skilled in the art may refer and which isincorporated herein by reference. Thus, according to an embodiment, theerythrocytes encapsulating the enzyme, are obtained by a methodcomprising the encapsulation of the active ingredient insideerythrocytes by lysis-resealing, the obtaining of a suspension or of apellet comprising erythrocytes incorporating the enzyme and a solutionwith an osmolality greater than or equal to 280 mOsmol/kg, in particularbetween about 280 and about 380 mOsmol/kg, preferably between about 290and about 330 mOsmol/kg, the incubation of the pellet or of thesuspension as such or after adding an incubation solution, at anosmolality greater than or equal to 280 mOsmol/kg, in particular betweenabout 280 and about 380 mOsmol/kg, preferably between about 290 andabout 330 mOsmol/kg. Incubation is notably carried out for a periodgreater than or equal to 30 minutes, in particular greater than or equalto 1 h. It is then proceeded with removal of the liquid medium of theincubated solution and the erythrocytes obtained are suspended in asolution allowing injection of the suspension into a patient, preferablya preservation solution allowing injection of the suspension into apatient. The indicated osmolality is that of the solution in which theerythrocytes are suspended or in a pellet at the relevant moment.

By “stabilized erythrocyte suspension”, is notably meant a suspensionhaving an extracellular hemoglobin content which remains less than orequal to 0.2 g/dl until its use in humans, the latter may intervenenotably from 1 to 72 hours after producing the erythrocyte batchincorporating the active ingredient.

By “ready-to-use stabilized erythrocyte suspension”, is meant thestabilized suspension in a solution allowing injection into a patient,notably in a preservation solution. Its hematocrit is generally equal toor greater than 35%, 40% or 45%.

By “erythrocyte pellet”, is meant a concentrate or concentration oferythrocytes collected after separating the erythrocytes of the liquidmedium in which they were suspended previously. The separation may beensured by filtration or by centrifugation. Centrifugation is the meansgenerally used for such a separation. A pellet comprises a certainproportion of liquid medium. Generally, the pellet has a hematocritcomprised between 70 and 85%.

By “incubation solution”, is meant the solution in which theerythrocytes encapsulating an active ingredient are present during theincubation step. The incubation may be accomplished over a large rangeof hematocrits, notably between 10 and 85% of hematocrit.

By “fragile erythrocytes”, are meant the erythrocytes stemming from theincorporation procedure which may, once suspended in a preservationsolution, be lysed when the suspension is preserved between 2 and 8° C.,notably after 1 to 72 h.

By “initial hematocrit”, is meant the hematocrit before cell loss due tolysis of the fragile erythrocytes during incubation.

The method may notably comprise the following steps:

(a) encapsulation of the enzyme inside erythrocytes, comprising theputting of the erythrocytes into contact with a hypotonic medium(allowing opening of pores in the membrane of the erythrocytes), thecontacting with the active ingredient (for allowing it to enter theerythrocytes), the resealing of the erythrocytes, notably by means of anisotonic or hypertonic medium, advantageously hypertonic,

(b) obtaining or preparing a suspension or pellet comprisingerythrocytes incorporating the enzyme and a solution with an osmolalitygreater than or equal to 280 mOsmol/kg, in particular between about 280and about 380 mOsmol/kg, preferably between about 290 and about 330mOsmol/kg,

(c) incubating the pellet or the suspension of step (b) as such or afteradding an incubation solution, at an osmolality greater than or equal to280 mOsmol/kg, in particular between about 280 and about 380 mOsmol/kg,preferably between about 290 and about 330 mOsmol/kg, for a periodgreater than or equal to 30 minutes, notably greater than or equal to 1h,

(d) removing the liquid medium of the incubated suspension of step (c),

(e) suspending the erythrocytes obtained under (d) into a solutionallowing injection of the suspension into a patient, preferably apreservation solution allowing injection of the suspension into apatient.

According to a first method, the step following the encapsulation bylysis-resealing, notably step (b), includes at least 1 washing cycle,preferably 2 or 3 washing cycles, by dilution of the obtained suspensionor pellet in the lysis-resealing step or step (a) in a solution, at anosmolality greater than equal to 280 mOsmol/kg, in particular betweenabout 280 and about 380 mOsmol/kg, preferably between about 290 andabout 330 mOsmol/kg, and then obtaining a pellet of erythrocytes or asuspension. This pellet or this suspension comprises erythrocytesincorporating the enzyme and a solution with an osmolality greater thanor equal to 280 mOsmol/kg, in particular between about 280 and about 380mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg. Thefollowing steps, e.g. (c), (d) and (e) are then applied.

According to a second method, in the lysis-resealing step or step (a),resealing of the erythrocytes by means of an isotonic or hypertonicmedium produces the suspension of erythrocytes which may then be subjectto incubation, e.g. the suspension of step (b), in a solution with anosmolality greater than or equal to 280 mOsmol/kg, in particular betweenabout 280 and about 380 mOsmol/kg, preferably between about 290 andabout 330 mOsmol/kg. In other words, the lysis-resealing step or step(a) includes a step for resealing the erythrocytes wherein the suspendederythrocytes encapsulating the enzyme are mixed with an isotonic orhypertonic resealing solution, advantageously hypertonic, producing asuspension of erythrocytes with an osmolality greater than or equal to280 mOsmol/kg, in particular between about 280 and about 380 mOsmol/kg,preferably between about 290 and about 330 mOsmol/kg. In this method,the incubation step or step (c) comprises incubation of the suspensionstemming from the resealing. The incubation is carried out for a periodgreater than or equal to 30 minutes, notably greater than or equal to 1h. The following steps, e.g. (d) and (e) are then applied.

The steps following the lysis-resealing, e.g. (b) to (e), are conductedunder conditions resulting in the lysis of fragile erythrocytes, or of amajority of them, notably more than 50, 60, 70, 80 or 90%, or more. Todo this, it is possible to act on the incubation period, the incubationtemperature and on the osmolality of the solution in which theerythrocytes are suspended. The higher the osmolality, the longer theincubation time may be. Thus the lower the osmolality, the shorter maybe the incubation in order to obtain the same effect. Also, the higherthe temperature, the shorter the incubation time may be, and vice versa.One or several washing cycles will then allow removal of cell debris andextracellular hemoglobin, as well as the extracellular enzyme.

According to the invention, a washing cycle comprises the dilution ofthe suspension or pellet of erythrocytes, and then the separationbetween the erythrocytes and the washing solution. Preferably, a washingstep comprises preferably 2 or 3 dilution-separation cycles. Theseparation may be achieved by any suitable means, such as filtration andcentrifugation. Centrifugation is preferred.

Incubation is not limited by the hematocrit of the suspension. In thisway, a suspension having an initial hematocrit generally comprisedbetween 10 and 85%, notably between 40 and 80% may be incubated. This israther referred to as a pellet from 70% and as a suspension below thisvalue.

The removal step or step (d) aims at removing the liquid portion of thesuspension or of the incubated pellet, in order to notably remove celldebris and the extracellular hemoglobin, as well as consequently theextracellular enzyme.

According to a first method for the removal step or step (d),separation, notably centrifugation is carried out, this being notablyapplicable to a suspension. This separation may be followed by one orseveral, for example 2 or 3, washing cycles, by dilution in an isotonicsolution, and then separation, notably by centrifugation.

According to a second method for the removal step or step (d), dilutionbefore separation notably centrifugation is carried out, this beingapplicable to a suspension or to a pellet. The dilution may notably becarried out with an isotonic washing solution or with a preservationsolution.

The final step or step (e) consists of preparing the final suspensionsuch that it may be administered to the patient, without any othertreatment.

According to a first method for this step, a dilution of the erythrocytepellet from the removal step or step (d) is carried out with theinjection solution, notably the preservation solution.

According to a second method for this step, one or several cycles forwashing the erythrocyte pellet stemming from the removal step or step(d) is carried out with the injection solution, notably the preservationsolution, by dilution followed by separation. After washing, theerythrocytes are re-suspended in the injection solution, notably thepreservation solution.

The method of the invention may further comprise one, several or thetotality of the following features:

-   -   the incubation step or step (c) is carried out at a temperature        comprised between about 2 and about 39° C., over sufficient time        for ensuring lysis of fragile erythrocytes;    -   the incubation step or step (c) is carried out at a low        temperature, notably comprised between about 2 and about 10° C.,        in particular between about 2 and about 8° C., and lasts for        about 1 h to about 72 h, notably from about 6 h to about 48 h,        preferably from about 19 h to about 30 h;    -   the incubation step or step (c) is conducted at a higher        temperature comprised between about 20 and about 39° C., notably        at room temperature (25° C.±5° C.) and lasts for about 30 min to        about 10 h, notably from about 1 h to about 6 h, preferably from        about 2 h to about 4 h; it is possible to operate at an even        higher temperature than room temperature, but this may have a        negative impact on the cell yield, P50 and/or the 2,3-DPG        content;    -   in the incubation step or step (c), the suspension is at an        initial hematocrit comprised between 10 and 85%, notably between        40 and 80%; a pellet from separation, having for example a        hematocrit between 70 and about 85%, or a diluted pellet having        a hematocrit comprised between about 40 and 70% may be        incubated;    -   the incubation step comprises stirring of the suspension;    -   the incubation step does not comprise any stirring;    -   as a solution for washing and/or incubation, a metered aqueous        NaCl solution is used for obtaining the desired osmolality; as        an example, a solution may thus comprise 0.9% of NaCl; this        solution may also comprise, notably in addition to NaCl,        glucose, notably glucose monohydrate, monosodium phosphate        dihydrate, disodium phosphate dodecahydrate; as an example, a        composition comprises: 0.9% of NaCl, 0.2% of glucose        monohydrate, 0.034% of monosodium phosphate dihydrate, 0.2% of        disodium phosphate dodecahydrate;    -   the washing in the final step or step (e) is carried out with        the preservation solution;    -   the osmolality of the solution (liquid portion) in the        ready-to-use suspension or which may be injected into the        patient is comprised between about 280 and about 380 mOsmol/kg,        preferably between about 290 and about 330 mOsmol/kg;    -   the hematocrit of the ready-to-use suspension or which may be        injected into the patient is equal to or greater than 35%, 40%        or 45%;    -   all the steps for washing and incubation are carried out with        the preservation solution;    -   the washing solution of step (b) and/or the washing solution of        step (e) and the preservation solution are of the same        composition and comprise compound(s) promoting preservation of        the erythrocytes;    -   the preservation solution (and the washing solution(s) or the        incubation solutions if necessary) is an aqueous solution        comprising NaCl, adenine and at least one compound from among        glucose, dextrose and mannitol;    -   the preservation solution (and the washing or incubation        solution(s) if necessary) comprises NaCl, adenine and dextrose,        preferably an AS3 medium;    -   the preservation solution (and the washing or incubation        solution(s), if necessary) comprise NaCl, adenine, glucose and        mannitol, preferably a SAG-Mannitol or ADsol medium.

The methods according to the invention notably comprise the followingstep:

(a) encapsulating the enzyme inside erythrocytes, comprising thecontacting with a hypotonic medium allowing opening of pores in themembrane of the erythrocytes, the contacting with the enzyme in order toallow its entry into the erythrocytes, the resealing of the erythrocytesby means of an isotonic or hypertonic medium. It should be noted thatthe enzyme may be present in the suspension of erythrocytes before thelysis of the latter, or further be added during lysis or after lysis,but always before resealing. In an embodiment of this step (a), themethod comprises the following sub-steps:

(a1) having a suspension of erythrocytes at a hematocrit equal to orgreater than 60 or 65%,

(a2) measuring the osmotic fragility of the erythrocytes in thissuspension,

(a3) a procedure for lysis and internalization of the activeingredient(s), comprising the passing of the erythrocyte suspension intoa dialysis device, notably a dialysis cartridge, counter to a lysissolution, adjusting the flow of the erythrocyte suspension or adjustingthe flow rate of the lysis solution or adjusting the osmolarity of thelysis solution, depending on the osmotic fragility measured under (a2),

(a4) a procedure for resealing the erythrocytes.

Definitions given herein above in the section “Arginine deiminase”,“Pharmaceutical compositions” or “suspension”, “Therapeutic use” and“dose”, such as “erythrocytes”, “hematocrit level”, “Arginine deiminase”apply mutatis mutandis to the section “Methods of encapsulation”.

Throughout the instant application, features described in one sectionare entirely applicable to other sections of the instant description.For instance, the description referring to “Arginine deiminase” as givenin the section “Arginine deiminase” is entirely applicable to thesection called “Pharmaceutical compositions or suspensions”, the sectioncalled “Therapeutic methods and uses”, the section “Therapeutic methodsand uses”, the section “Methods of encapsulation” and the section“dose”.

Throughout the instant application, the term “and/or” is a grammaticalconjunction that is to be interpreted as encompassing that one or moreof the cases it connects may occur. For example, the wording“low-protein diet and/or nitrogen scavenger drugs” in the phrase“outcomes are usually poor with a low-protein diet and/or nitrogenscavenger drugs” indicates that outcomes are poor for individualstreated with either a low-protein diet or nitrogen scavenger drugs or acombination thereof, i.e. a low-protein diet and nitrogen scavengerdrugs.

Throughout the instant application, the term “comprising” is to beinterpreted as encompassing all specifically mentioned features as welloptional, additional, unspecified ones. As used herein, the use of theterm “comprising” also discloses the embodiment wherein no featuresother than the specifically mentioned features are present (i.e.“consisting of”). Furthermore the indefinite article “a” or “an” doesnot exclude a plurality. The mere fact that certain measures are recitedin mutually different dependent claims does not indicate that acombination of these measures cannot be used to advantage.

The invention will now be described in more details with reference tothe following figures and examples. All literature and patent documentscited herein are hereby incorporated by reference. While the inventionhas been illustrated and described in detail in the foregoingdescription, the examples are to be considered illustrative or exemplaryand not restrictive.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the amino acid sequence of full-length argininedeiminase from Mycoplasma arginini as available from the NCBI databaseunder accession number WP_004416214.1

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the urea cycle showing the role ofthe enzymes (NAGS, CPS1, ASS1, ASL, ARG1, OTC) and the transporters(ORNT1 and citrin) in conversion of ammonia nitrogen in urea.

FIG. 2: Graph representing the Pharmacokinetics (PK) of CFSE-labelederythrocytes encapsulating ADI. ERY-ADI 6 product is obtained bylysis-resealing of a suspension containing 5 mg/ml of ADI. Fluorescentlabeling of the products (CFSE) allows traceability of the erythrocytesin vivo. The product injected intravenously to the mice C57BL/6 (8ml/kg) has excellent stability with a half-life estimated between 18 and22 days after their administration.

FIG. 3: Graph representing the Pharmacodynamics of erythrocytesencapsulating ADI over 16 days. The product ERY-ADI 6 is obtained bylysis-resealing of a suspension containing 5 mg/ml of ADI. The productis administered intravenously (IV) to C57BL/6 mice (8 ml/kg). The plasmaL-arginine level is measured by HPLC-MS-MS. The L-Arginine level inuntreated C57BL/6 mice was evaluated to be between 75 and 125 μM. Theproduct ERY-ADI 6 leads to a rapid depletion 15 min after administrationreducing the L-Arginine level to 0 μM for 13 days. On day 16, two out ofthree mice displayed a complete plasma L-arginine depletion. PlasmaL-arginine of the third mouse was 13 μM. The average plasma L-argininelevel for this group of mice at Day 16 is 4.33±7.51 μM.

FIG. 4: Graph representing the blood L-Arginine level inarginase-deficient mice after one single intravenous administration oferythrocytes encapsulating ADI. The products ERY-ADI 4 is obtained bylysis-resealing of a suspension containing 4.5 mg/mL of ADI. As acontrol, a product named pRBC (Processed Red Blood Cells) has beenobtained by lysis-resealing process without ADI enzyme (mock-loadederythrocytes). On day 0, the two products are administered intravenouslyto arginase-deficient mice (4 or 8 mL/kg for ERY-ADI 4 and 8 mL/kg forpRBC). Blood sampling is performed on Day 0 (before ERY-ADI 4 or pRBCadministrations) Day 1 and Day 3 for all mice. When ERY-ADI 4 wasadministered at 4 mL/kg, blood L-Arginine was lowered by 60% and by 7%,1 and 3 days after injection, respectively. When double dose volume ofERY-ADI 4 was administered (8 mL/kg), the efficacy of erythrocytesencapsulating ADI on pathological blood L-Arginine level is spectacular;the following day after injection, the L-Arginine level is more that10-fold lower than the L-Arginine concentration baseline of this mousemodel (35±22 μM vs 452±45 μM corresponding to 92% blood depletion).Three (3) days after administration of ERY-ADI 4 product, bloodL-Arginine level is still 4 times lower than the pathobiochemical level(119±57 μM vs 452±45 μM corresponding to 73% blood depletion). However,when mock-loaded erythrocytes (pRBC) were administered, no bloodL-Arginine depletion has been observed. On the contrary, bloodL-Arginine level still increased, demonstrating that mock-loadederythrocytes had no effect on the biochemical course of the disease.

FIG. 5: Graph representing the serum Ammonia level in arginase-deficientmice after one single intravenous administration of erythrocytesencapsulating ADI. The products ERY-ADI 4 is obtained by lysis-resealingof a suspension containing 4.5 mg/mL of ADI. As a control, a productnamed pRBC (Processed Red Blood Cells) has been obtained bylysis-resealing process without ADI enzyme (mock-loaded erythrocytes).On day 0, the two products are administered intravenously toarginase-deficient mice (4 or 8 mL/kg for ERY-ADI 4 and 8 mL/kg forpRBC). Blood sampling is performed on Day 0 (before ERY-ADI 4 or pRBCadministrations) and Day 3 for all mice to assay serum ammonia. SerumAmmonia was analyzed as the conversion of L-Arginine by ADI results inthe production of Citrulline and Ammonia. No notable changes in ammonialevels have been observed when the mice were treated with erythrocytesencapsulating ADI or mock-loaded erythrocytes.

FIG. 6: Graph representing the blood L-arginine levels inarginase-deficient mice after administrations of ERY-ADI once (Group 2),twice (Group 3) or the free form of ADI enzyme (Group 4).

FIG. 7: Graph representing the blood L-arginine levels inarginase-deficient mice after administrations of Mock RBC (Group 1),ERY-ADI once (Group 2) and twice (Group 3).

EXAMPLES Example 1. Method for Obtaining and Characterizing ArginineDeiminase (ADI)

Production of the strain and isolation of a hyper-producing clone: Thenatural sequence of ADI from Mycoplasma arginini (GenBank: X54141) wasoptimized by modifying some codons because genetic codes between E. coliand M. arginini are different (a new plasmid was created codedC1124-ADM-02). The purification process has been described in Misawa andcoll. (Misawa, S. et al, 1994, J. of Biotechno. 36, 1994, 145-155) withsome modifications, i.e., the bacterial production HMS174 (DE3) T1Rstrain has been used instead of the JM101 strain. Other modificationsare described below.

Fermentation:

The production was achieved in a fermenter with FED_Coli_9 batch medium,with stirring, controlled pressure and pH from the pre-culture 2 at anoptical density of 0.05. The growth phase (at 37° C.) took place untilan optical density (600 nm) of 100 was obtained and the expressioninduction was achieved at 32° C. by adding 1 mM IPTG into the culturemedium. The cell sediment was harvested 26-27 h after induction in twophases: the cell broth was concentrated 5-10 times after passing over a500 kDa hollow fiber and then the cell pellet was recovered bycentrifugation at 15900×g and then stored at −20° C.

Purification:

ADI was produced as inclusion bodies (IB). The cell pellets weresuspended in a lysis buffer to disrupt the cells. Then the disruptedcells were washed to collect the IBs and the IBs were stored at −20° C.

The purification of ADI started by thawing the IB pellet in a buffercomposed of 50 mM TRIS base pH 8.5, 6M Guanidinium Hydrochloride, 10 mMDithiothreitol. The solubilization was achieved with an incubation timeof 1 h at 37±2° C. After clarification, the refolding step took placefor 40-45 hours at room temperature in a buffer composed of 3 mMmonopotassium phosphate (KH2PO4), 7 mM dipotassium phosphate (K2HPO4) pH7,35. After a second clarification step, the medium was loaded onto aQ-sepharose column. Elution was performed with 250 and 500 mM NaCl andthe elution fraction was submitted to a tangential flow filtration(TFF). Two polishing steps (using Sartobind Q column) and two TFF stepscomplete the purification of ADI. A final 0.2 μM filtration wasperformed before storage of ADI at −20° C.

Characterization: The specific activity of the enzyme was determined bymeasuring the produced Citrulline as described in example 2. The proteincontent was determined by reading absorbance at 280 nM. The purity wasdetermined by SDS-PAGE. The osmolarity was measured with an osmometer(Micro-Osmometer Loser Type 15). The main characteristics of oneproduced batch of ADI are summarized herein below in table 2.

TABLE 2 Main characteristics of one produced batch of ADI ADI of M.arginini Formulation Liquid phase frozen at −80° C. Characteristics: 323mOsm/Kg - 16,65 mg/mL 50 mM phosphate de sodium pH 6.5, Sucrose 40mg/mL, Lysine 40 mM Specific activity ~47 U/mg Purity 97.5%

Example 2. ADI Specific Activity Assay Using Citrulline Measurement

This assay is based on a 2-step reaction (Boyde and Rahmatullah, 1980,Analytical Biochemistry, vol 107, p 424-431):

-   -   First, L-Arginine is converted into citrulline and ammonia by        ADI    -   Second, in presence of diacetyl monoxime, iron (Ill) chloride,        thiosemicarbazide, sulfuric and phosphoric acids, citrulline is        converted into a colored chromophore readable at 530 nm.

A L-citrulline standard curve is prepared to determine the ADI enzymaticactivity of all assay samples, by reading the absorbance at 530 nm.Specific activity (U/mg) is calculated using the enzymatic activity(U/mL) and the protein content (mg).

Example 3. Encapsulation of ADI in Murine Erythrocytes

Whole blood of C57BL/6 mice (Charles River) was centrifuged at 1000×g,for 10 min, at 4° C. to remove the plasma and buffy coat. Theerythrocytes were washed three times with 0.9% NaCl (v:v). The frozenADI was thawed and added to the erythrocyte suspension in order toobtain a final suspension with a hematocrit of 65%, containing aninitial concentration of ADI of 2 to 7 mg/mL.

The suspension was then loaded on a hemodialyzer at a flow rate of 120ml/h and dialyzed against a hypotonic solution (40-50 mOsmol/kg) at aflow rate of 15 ml/min as a counter-current. The suspension was thenresealed with a hypertonic solution (1 600-2 100 mOsmol/kg) and thenincubated for 30 min at 37° C. After three washes in 0.9% NaCl, 0.2%glucose, the suspension was taken up in a preservation solution AS3supplemented with 20% decomplemented plasma.

The obtained products are characterized at the time point D0 (within the2 h following their preparation) and at time point D1 (i.e. after ˜18h-24 h of storage at 2-8° C.). The hematologic characteristics areobtained with a veterinary automaton (Sysmex, PocH-100iV).

Results:

ADI activity in the finished products was assayed with the methoddescribed in example 4 against an external calibration range of ADI inaqueous solution. These results show that ADI activity in the finishedproducts increases with the amount of enzyme introduced into the RBC andthat it is easily possible to encapsulate up to 2 mg of ADI per ml offinished product while maintaining good stability. The maincharacteristics of 6 different batches of ERY-ADI murine final products(ERY-ADI-1 to 6) are given herein below in Table 3.

TABLE 3 Main characteristics of 6 ERY-ADI murine final products asmeasured at the time point D0 (2 h following the preparation) ERY- ERY-ERY- ERY- ERY- ERY- Batches ADI-1 ADI-2 ADI-3 ADI-4 ADI-5 ADI-6Hematological Hematocrit (%) 51.1 51.1 50.8 51.3 51.1 51.3 parametersCorpuscle volume (fl) 39.5 40.1 40.6 41.3 40.0 38.60 Corpusclehemoglobin (g/dl) 26.6 26.3 25.3 26.7 28.2 25.7 Total hemoglobin (g/dl)14.8 14.4 14.0 14.8 15.6 13.8 Extracellular Hb (g/dl) 0.3 0.4 0.3 0.80.3 0.3 ADI ADI concentration 3 4.5 4.5 4.5 3.5 5 parameters beforeprocess (mg/mL) Intra-erythrocyte concentration of 1.15 1.56 1.90 1.131.37 2.70 ADI (mg/ml of RBC-100% Ht) Extracellular activity (%) 4.8 5.74.8 6 4.0 4.1 Intracellular activity (%) 95.2 94.3 95.2 94 96.0 95.9Encapsulation yield of ADI (%) 38 35 42 25 39 54

Example 4. Assay of Encapsulated ADI in the Erythrocytes

The assay of the ADI activity entrapped in red blood cells and in thesupernatants, is based on a measurement of NH₃ produced by ADI fromL-Arginine. The NH₃ ions were assayed indirectly by enzymatic action ofglutamate dehydrogenase (GLDH) according to the kit marketed by RocheDiagnostics (11877984).

Example 5. Pharmacokinetics of Erythrocytes Encapsulating ADI in C57BL/6Mice

The murine product ERY-ADI 6 was labeled with CFSE (fluorescent) andadministered intravenously into C57BL/6 mice. At each time points (D0+15min, D0+6 h, D1, D2, D5, D9, D13 and D16), 3 mice were sacrificed andthe blood was collected on a lithium heparinate tube kept at +4° C. awayfrom light for determining the pharmacokinetics. The proportion of redblood cells labeled with CFSE in the whole blood was determined by aflow cytometry method. Five microliters of whole blood were diluted in 1ml of PBS 0.5% BSA and each sample was passed in triplicate (counting of10,000 cells in FL-1; cytometer FC500, Beckman Coulter). The evaluationof the survival of red blood cells loaded with ADI was obtained byadding the proportion of erythrocytes loaded with ADI labeled with CFSEat different time points to the proportion of erythrocytes loaded withADI labeled with CFSE at T0+15 min (100% control). The differentobtained percentages for each time are indicated in the graph depictedin FIG. 2 thus illustrating the proportion of erythrocytes loaded withADI in circulation versus time.

Based on half-life calculation, CFSE-labeled erythrocytes encapsulatingADI have an estimated half-life comprised between 18 and 22 days.

Example 6. Pharmacodynamics of Erythrocytes Encapsulating ADI in C57BL/6Mice

The product ERY-ADI 6 of erythrocytes encapsulating ADI enzyme wasinjected intravenously to C57BL/6 mice at a dose of 8 ml/kg. At eachtime points (D0+15 min, D0+6 h, D1, D2, D5, D9, D13 and D16), 3 mice aresacrificed and the blood is collected on lithium heparinate tubes storedat 4° C. for the determination of plasma L-Arginine levels.

As shown in FIG. 3 a complete plasma L-Arginine depletion is observedfor 13 days immediately (i.e. 15 minutes) after administration oferythrocyte encapsulating ADI. At the last time point of the study (i.e.16 days), 2 mice out of 3 still displayed a complete plasma L-Argininedepletion. Plasma L-Arginine level of the third mouse was 13 μM, muchlower than the physiological plasma L-Arginine concentration in thisstudy (100±25 μM).

Example 7. Administration of Erythrocytes Encapsulating ADI toArginase-Deficient Mice (1^(st) Study)

An in vivo study was set up with an arginase-deficient mouse model (Fora complete description see Sin et al, 2013, PLOS One, vol. 8 (11)).These mice are devoid of arginase 1 activity and exhibit severepathobiochemical aspects of hyperargininemia commonly seen in humans.Hyperargininemia (or Arginase deficiency) is triggered by 5 injectionsof tamoxifen. Blood arginine concentrations start to rise few days afterthe last tamoxifen injection. To demonstrate an efficacy of murineproduct ERY-ADI 4 to decrease blood L-Arginine level in this mousemodel, ERY-ADI 4 (4 and 8 mL/kg) and mock-loaded erythrocytes (8 mL/kg)were intravenously injected to 15 arginase-deficient mice 7 days afterthe last tamoxifen injection. Blood was collected the day (D1) and twodays later (D3) after administration of the three products. Results ofthe in vivo study are presented in FIGS. 4 and 5 and in Table 4 below.

TABLE 4 Summary of the results of the in vivo study of ERY-ADI-4 in anarginase-deficient mouse model. Indicated are the % depletion of bloodArginine levels. Negative numbers indicate an increase of Argininelevels. % depletion Mock loaded ERY-ADI 4 ERY-ADI 4 (Blood L-Arg)erythrocytes (8 mL/kg) (4 mL/kg) (8 mL/kg) D1 −10% 60% 92% D3 −30% 7%73%

As shown in this table and in FIG. 4, erythrocytes encapsulating ADI,when administered at a dose volume of 4 mL/kg, decreased bloodL-Arginine by 60% and by 7%, 1 and 3 days after injection, respectively.When double dose volume was administered (8 mL/kg), the efficacy oferythrocytes encapsulating ADI on pathological blood L-Arginine level isspectacular; the following day after injection, the L-Arginine level ismore that 10-fold lower than the L-Arginine concentration baseline ofthis mouse model (35±22 μM vs 452±45 μM corresponding to 92% blooddepletion). Three (3) days after administration of ERY-ADI 4 product,blood L-Arginine level is still 4 times lower than the pathobiochemicallevel (119±57 μM vs 452±45 μM corresponding to 73% blood depletion).However, when mock-loaded erythrocytes were administered, no bloodL-Arginine depletion has been observed. On the contrary, bloodL-Arginine level still increased, demonstrating that mock-loadederythrocytes had no effect on the biochemical course of the disease.

Serum Ammonia was analyzed at the same time as the conversion ofL-Arginine by ADI results in the production of Citrulline and Ammonia.As shown in FIG. 5, no notable changes in ammonia levels have beenobserved when the mice were treated with erythrocytes encapsulating ADIor mock-loaded erythrocytes.

Example 8. Administration of Erythrocytes Encapsulating ADI toArginase-Deficient Mice (Second Study)

To confirm the efficacy of murine product ERY-ADI to decrease bloodL-Arginine level in this mouse model, a second study was set up on micetreated with tamoxifen in accordance with example 7. ERY-ADI product wasintravenously injected 3 days after the last tamoxifen injection. One ortwo intravenous administration(s) of ERY-ADI (at 8 mL/kg) was scheduled(Groups 2 and 3 respectively). A control group was administered the freeform of ADI enzyme (Group 4). A second control group composed of micebearing arginase activity was part of the study (Group 1).

Blood was collected the day (D3) and one week later (Day 10). The day ofsacrifice, mice blood was collected too (Day 13).

Results of the second in vivo study are presented in FIG. 6.

First, blood arginine levels were lower in this second study because wechanged the injections schedule related to the tamoxifen injections. Inthis study, the administration of ERY-ADI was planned 3 days instead of7 days after last tamoxifen injection, resulting in a lower bloodarginine level baseline.

As shown in Figure. 6, erythrocytes encapsulating ADI, when administeredat a dose volume of 8 mL/kg, decreased blood L-Arginine by 81% and 77%compared to baseline levels for groups 2 and 3 respectively at Day 10(i.e. 7 days after administration). The second administration of ERY-ADIwas scheduled after the blood collection on day 10. Ten (10) days afteradministration, depletion of blood arginine is still very important withsome percentages of depletion of 82% and 68% for group 2 and group 3respectively compared to baseline levels. In contrast no blood argininedepletion was observed in mice injected with free form of ADI at Day 10(Group 4). On the contrary the blood arginine concentration reached aconcentration of 349 μM reflecting the blood arginine level increase inthe model of arginase-deficient mice. No measurement of blood L-Argininelevel could be performed at Day 13 for group 4 since all animals diedfurther to the 2^(nd) injection with free form of ADI.

This second study confirmed the results observed with the first studywith some additional information about the pharmacodynamics of theERY-ADI product when injected to arginase-deficient mice. One singleadministration allows a blood arginine depletion for at least 10 daysand a second injection of ERY-ADI product did not result in any sideeffects on this mouse model.

Example 9. Administration of Erythrocytes Encapsulating ADI toArginase-Deficient Mice (Third Study)

As no survival was observed when ERY-ADI administration was performed 3days after the last tamoxifen injection, the third study was designedwith an important change in test item administration schedule. ERY-ADIwas intravenously injected the day before the first tamoxifen injection(Day 0). In this study, the last tamoxifen injection was carried out onDay 5. One or two intravenous administration(s) of ERY-ADI was scheduled(Groups 2 and 3, respectively). Group 1 was a control group composed ofArginase-deficient mice administered the mock RBC (i.e. processed RBCwith no ADI). Blood was collected on Day 0, Day 11, Day 14 (beforesecond administration of ERY-ADI) and when mice were sacrificed forexcess body weight loss (identified as “end of study”).

As shown in FIG. 7, erythrocytes encapsulating ADI, when administered ata dose volume of 8 mL/kg, decreased blood L-Arginine by 71.4% and 78.7%compared to group 1 levels for groups 2 and 3 respectively at Day 11(i.e. 11 days after administration). At Day 14, before the secondadministration of ERY-ADI (for group 3 only), whole blood arginine wasstill depleted by 27.3% and 29.3% for groups 2 and 3 respectively,compared to group 1. In contrast, no whole blood arginine depletion wasobserved in mice injected with mock RBC (Group 1). In this group, thewhole blood arginine concentration reached a concentration of 844 μM atthe end of the study reflecting a whole blood arginine level increase inthis arginase-deficient mouse model.

When ERY-ADI was reinjected at Day 14 (group 3), whole blood argininelevel was measured few days later, at the time of sacrifice for ethicalreasons (identified as “end of study”). The blood arginine was stilldepleted following the second administration of ERY-ADI, butarginase-deficient mice has to be sacrificed due to significant bodyweight loss.

This third study confirmed the results observed with the first twostudies with some additional information about the pharmacodynamics ofthe ERY-ADI product when injected into arginase-deficient mice. A singleadministration yielded a blood arginine depletion for at least 11 daysin this mouse model and a second injection of ERY-ADI yielded asustained blood arginine depletion for at least 17 days (lifespan of thefirst mouse that had been sacrificed for ethical reasons).

However, due to the severity of this mouse model, no survival has beenobserved beyond 15 days after last tamoxifen injection, whatever thenumber of ERY-ADI administrations performed.

Example 10: Production of Human Red Blood Cells Encapsulating ArginineDeiminase

A pouch of leucocyte-depleted human RBCs (provided by the “Etablissementfrançais du sang”) was subjected to three washes with 0.9% NaCl. TheArginine Deiminase (ADI) solution was gently thawed and added to the RBCsuspension to obtain a final concentration with a hematocrit of 60%containing 3 or 5 mg/mL of ADI. The suspension was homogenized andloaded on a hemodialyzer at a flow rate of 90 mL/h and dialyzed againsta hypotonic solution at 30 mOsmol/kg. The suspension was then resealedwith a hypertonic solution and then incubated for 30 minutes a 30° C.After 3 washes in 0.9% NaCl, 0.2% Glucose, the suspension was taken upin a preservative solution AS3 (NaCl, NaH₂PO₄, Citric acid, Na-citrate,adenine and glucose. Osmolality is 288 mOs/kg and pH 5,88). The productsobtained were characterized at Day 0, Day 1 and Day x. The hematologiccharacteristics were obtained with a veterinary automat (Sysmex,PocH-100iV).

It is important to note that no magnesium, iron or other enzymeco-factor was (or need be) added to the disclosed RBC-encapsulatedarginine deiminase (ADI) compositions and suspensions. Unlikenon-encapsulated ADI preparations, which may make use of magnesium orother co-factors present in a subject's bloodstream, the ADI of thepresent disclosure is generally limited to the contents of the RBCs. Tosolve this problem, Applicants specifically selected aco-factor-independent ADI (e.g. M. arginini) to ensure long-lasting invivo ADI activity, without the need to supplement the products withmagnesium or some other co-factor. Applicants envision that otherco-factor-independent ADI may be used effectively in the practice of thedisclosed invention.

As used herein, “Co-factor-independent ADI” means an ADI that does notdepend upon enzyme co-factors such as vitamins, pro-vitamins, vitaminprecursors, or metal ions (e.g. magnesium, iron, manganese, etc.). Asused herein, “magnesium-independent ADI” means an ADI that does notdepend upon magnesium to support its enzymatic activity.

Results. The hematologic and biochemical characteristics of 6 finishedproducts at Day 0 (day of manufacturing) are compiled in Table 5 below:three were manufactured with an ADI concentration of 3 mg/mL and threewith an ADI concentration of 5 g/mL, expressed with respect to the RBCsuspension before dialysis. All the ERY-ADI products were prepared withthe same batch of ADI. In vitro stability was assessed on Day 0, Day 1(Table 6) and Day 7 (Table 7).

TABLE 5 Hematologic and biochemical characteristics of ERY-ADI I to VI(Day 0) ADI 3 mg/mL ADI 5 mg/mL ERY- ERY- ERY- ERY- ERY- ERY- Day 0parameters ADI I ADI II ADI III ADI IV ADI V ADI VI Hemato- Hematocrit(%) 48.2 50.8 48.0 50.4 48.0 49.2 Logical Corpuscular volume (fL) 87.582.9 85.7 85.3 83.3 86.9 data Corpuscular 27.6 28.6 28.6 28.1 29.4 27.9hemoglobin (g/dL) RBC count (10⁶/μL) 5.52 6.12 5.60 5.92 5.78 5.66 TotalHemoglobin (g/dL) 13.4 14.4 13.8 14.2 14.2 13.8 Extracellular Hb (g/dL)0.1 0.1 0.1 0.1 0.1 0.1 ADI Intra-erythrocyte 0.88 0.94 1.01 1.55 1.361.46 concentration of ADI (mg/mL of RBC) Intra-erythrocyte 42.1 44.948.3 74.1 65.0 69.8 activity of ADI (U/mL) Extracellular activity (%)1.2 0.0 3.0 0.6 0.8 4.0 Intracellular activity (%) 98.8 100.0 97.0 99.499.2 96.0 Entrapment yield of ADI (%) 29.4 31.5 33.7 31.0 27.3 29.3

Independent of the ADI concentration added before entrapment, theentrapment yield was very reproducible (from 27.3 to 33.7%).

TABLE 6 Hematologic and biochemical characteristics of ERY-ADI Ito VI(Day 1) ADI 3 mg/mL ADI 5 mg/mL ERY- ERY- ERY- ERY- ERY- ERY- Day 1parameters ADI I ADI II ADI III ADI IV ADI V ADI VI Hemato- Hematocrit(%) 46.6 51.4 47.8 49.4 49.0 46.4 Logical Corpuscular volume (fL) 85.581.6 83.8 83.8 81.4 84.7 Data Corpuscular 28.8 30.3 29.1 28.3 29.6 29.1hemoglobin (g/dL) RBC count (10⁶/μL) 5.46 6.28 5.70 5.90 6.02 5.48 TotalHemoglobin (g/dL) 13.4 15.6 14.0 14.0 14.4 13.4 Extracellular Hb (g/dL)0.1 0.1 0.2 0.1 0.1 0.2 ADI Intra-erythrocyte 0.89 0.83 1.00 1.50 1.221.86 concentration of ADI (mg/mL of RBC) Intra-erythrocyte 42.5 39.747.8 71.7 58.3 88.9 activity of ADI (U/mL) Extracellular activity (%)1.3 1.1 6.0 1.3 1.7 5.1 Intracellular activity (%) 98.7 98.9 94.0 98.798.3 94.9

TABLE 7 Hematologic and biochemical characteristics of ERY-ADI I to VI(Day 7) ADI 3 mg/mL ADI 5 mg/mL ERY- ERY- ERY- ERY- ERY- ERY- Day 7parameters ADI I ADI II ADI III ADI IV ADI V ADI VI Hemato- Hematocrit(%) 47.6 51.4 50.4 50.4 48.0 47.6 Logical Corpuscular volume (fL) 86.482.8 86.6 85.3 82.6 87.6 data Corpuscular 27.4 28.8 27.3 28.2 29.6 27.6hemoglobin (g/dL) RBC count (10⁶/μL) 5.52 6.22 5.82 5.90 5.82 5.44 TotalHemoglobin (g/dL) 13.0 14.8 13.8 14.2 14.2 13.2 Extracellular Hb (g/dL)0.3 0.5 0.5 0.4 0.5 0.5 ADI Intra-erythrocyte 0.85 0.80 0.91 1.35 1.221.54 concentration of ADI (mg/mL of RBC) Intra-erythrocyte 40.6 38.243.5 64.5 58.3 73.6 activity of ADI (U/mL) Extracellular activity (%)3.7 5.2 11.5 4.2 3.7 12.7 Intracellular activity (%) 96.3 94.9 88.5 95.896.3 87.3

Entrapment of Arginine Deiminase in human red blood cells proved to be avery reproducible process. The main parameters were stable between Day 0and Day 1 (e.g. extracellular hemoglobin, intracellular andextracellular enzyme activity, hematocrit, corpuscular volume). At Day7, the measured parameters indicated that the ERY-ADI product is verystable in vitro, independently of the ADI concentration added before theentrapment process.

Conclusion. Arginine Deiminase (ADI; EC number 3.5.3.6) entrapped in redblood cells (RBC), was obtained using the Erytech's proprietary ERYCAPS®technology platform. The entrapment of therapeutic enzymes into redblood cells can provide effective, long-acting therapeutic activity withreduced toxicity.

Entrapment of Arginine Deiminase inside Red Blood Cells (ERY-ADIproduct), greatly improves the pharmacological properties of the enzyme.In healthy mice, plasma L-arginine depletion was complete within 15minutes after administration and was sustained for 13 days.

When injected to Arginase-deficient mice, ERY-ADI demonstrated aspectacular efficacy on the very high blood L-arginine concentrationsdisplayed by these mice. Indeed, 24 h after administration, bloodL-arginine concentration was reduced by 52 and 92% when 4 or 8 mL/kgwere injected, respectively. Three days after administration, bloodL-arginine level remained reduced by 19 and 73%. Moreover, despite theproduction of ammonia by Arginine Deiminase, the serum level stayedcomparable to the mock-loaded RBC control.

These results were confirmed in a second study wherein a singleadministration yielded a blood arginine depletion for at least 10 daysand a second injection of ERY-ADI was well tolerated byarginase-deficient mice.

Entrapment of Arginine Deiminase was successfully performed in human RedBlood Cells with a good reproducibility and in vitro stability.

Based on these results, ERY-ADI is envisioned to be capable ofcounteracting the primary biochemical defect of the rare geneticdisorder of Arginase deficiency.

The invention will now be described by the following numberedparagraphs:

1. A pharmaceutical composition comprising arginine deiminase (ADI)encapsulated into erythrocytes and optionally a pharmaceuticallyacceptable vehicle for its use in treating arginase-1 deficiency,preferably wherein the composition is capable of reducing pathologicalplasma or whole blood arginine levels in a patient or subject sufferingfrom Arginase 1 (Arg1) deficiency to normal physiological or near-normalphysiological plasma or whole blood arginine levels, more preferablywherein the composition is capable of reducing the pathological levelsof arginine by at least about 20, 30, 40, 50, 60, 70 or about 80% for aperiod of at least about 6, 7, 8, 9, 10 or 11 days post administrationof a single dose of the composition;

optionally wherein the ADI is any one, any combination, or all of thefollowing: co-factor-independent, magnesium-independent,iron-independent, vitamin-independent, pro-vitamin-independent; and

optionally wherein the composition is characterized by any or all of thefollowing ranges of values across the indicated parameters:

Hematocrit (%): about 48 to about 51;

Corpuscular volume (fL): about 82 to about 88;

Corpuscular hemoglobin (g/dL): about 27 to about 30;

RBC count (106/μL): about 5.5 to about 6.2;

Total Hemoglobin (g/dL): about 13.0 to about 14.5;

Extracellular Hb (g/dL): about 0.1 to about 0.2;

Intra-erythrocyte concentration of ADI (mg/mL of RBC): about 0.8 toabout 1.6;

Intra-erythrocyte activity of ADI (U/mL): about 42 to about 49 or about65 to about 74;

Extracellular activity (%): about 0.0 to about 4.0;

Intracellular activity (%): about 96 to about 100; and/or

Entrapment yield of ADI (%): about 27 to about 34.

2. The pharmaceutical composition for the use according to paragraph 1,wherein said composition is a suspension having an osmolarity of between270 and 350 mOsm/1.

3. The pharmaceutical composition for the use according to paragraph 1or 2, wherein the pharmaceutically acceptable vehicle is a preservativesolution comprising NaCl and Adenine.

4. The pharmaceutical composition for the use according to any one ofparagraphs 1 to 3, wherein said ADI is from M. arginini.

5. The pharmaceutical composition for the use according to any one ofparagraphs 1 to 4, wherein the ADI comprises the amino acid sequence ofSEQ ID NO: 1 or a variant or fragment thereof.

6. The pharmaceutical composition for the use according to paragraphs 5,wherein said variant comprises an amino acid sequence that is at least80% identical to the amino acid sequence SEQ ID NO: 1.

7. The pharmaceutical composition for the use according to paragraphs 5or 6, wherein said variant or fragment retains the biological activityof the ADI having the amino acid sequence of SEQ ID NO: 1.

8. The pharmaceutical composition for the use according to any one ofparagraphs 1 to 7, wherein the concentration of encapsulated ADI is from0.1 to 7 mg/ml.

9. The pharmaceutical composition for the use according to any one ofparagraphs 1 to 8, wherein the pharmaceutical composition is packaged ina dose having a volume from 10 to 250 ml.

10. The pharmaceutical composition for the use according to paragraph 9,wherein the amount of ADI encapsulated in one dose for a patient is from0.01 mg/kg to 500 mg/kg of encapsulated ADI per kg body weight of saidpatient.

11. A suspension of erythrocytes encapsulating ADI from M. arginini.

12. The suspension of paragraph 11, wherein said ADI comprises the aminoacid sequence of SEQ ID NO: 1 or a variant or fragment thereof.

13. The suspension of paragraph 12, wherein said variant comprises anamino acid sequence that is at least 80% identical to the amino acidsequence SEQ ID NO: 1.

14. A pharmaceutical composition comprising a suspension according toany one of paragraphs 11 to 13, for its use in treating arginase-1deficiency or arginine-dependent cancers, treating or preventing ofseptic shock, inhibiting angiogenesis and treating angiogenesisassociated diseases.

15. A method for treating arginase-1 deficiency or arginine-dependentcancers, treating or preventing of septic shock, inhibiting angiogenesisand treating angiogenesis associated diseases comprising administeringto a patient or subject in need thereof the composition or suspension ofany one of paragraphs 1 to 14.

16. A method for reducing pathological plasma arginine levels to normalphysiological plasma arginine levels in a patient or subject chronicallysuffering from said pathological plasma arginine levels, comprising thestep of administering more than one dose of the composition orsuspension of any one of paragraphs 1 to 14.

17. The method of paragraph 16, wherein the pathological plasma argininelevels are in excess of about 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590 or 600 μM.

18. The method of paragraph 16 or 17, wherein the patient or subject hasa known Arg1 mutation or exhibits less than about 5% arginase activityor substantially no arginase activity.

19. The method of paragraph 16 or 17, wherein the pathological plasmaarginine levels are at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or more fold higher than healthy subjects not suffering frompathological plasma arginine levels.

20. The method of any one of paragraphs 16 to 19, wherein one dose ofthe composition or suspension is sufficient to maintainnon-pathological, normal physiological plasma arginine levels for atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks.

21. The method of any one of paragraphs 16 to 20, wherein thecomposition or suspension only needs to be administered once every 2weeks or once every 4 weeks to maintain physiological plasma argininelevels.

The invention will now be described in the following non-limiting set ofClaims.

1.-14. (canceled)
 15. A pharmaceutically acceptable suspension oferythrocytes encapsulating a therapeutically effective amount of amagnesium-independent arginine deiminase (ADI), wherein whenadministered to a patient in need thereof, the suspension oferythrocytes delivers to said patient a sustained reduction in bloodarginine levels for at least 10 days post administration.
 16. Thesuspension of claim 15, further comprising a preservative solution forerythrocytes.
 17. The suspension of claim 15, wherein the ADI is a fulllength ADI, or a variant or fragment thereof retaining substantially thesame enzymatic activity and magnesium independence as the ADI having theamino acid sequence as set forth in SEQ ID NO:
 1. 18. The suspension ofclaim 15, wherein the ADI comprises an amino acid sequence that is atleast 80% identical to the amino acid sequence as set forth in sequenceSEQ ID NO:
 1. 19. The suspension of claim 18, wherein the ADI comprisesthe amino acid sequence as set forth in SEQ ID NO:
 1. 20. The suspensionof claim 15, wherein the concentration of the ADI enzymatic activitycomprises about 1 to about 400 U/ml, about 5 to about 400 U/ml, or about5 to about 350 U/ml, and/or wherein the amount of ADI comprises about0.5 to about 6.5 mg/ml, about 1 to 6 mg/ml, about 1 to about 5 mg/ml,about 1 to about 4 mg/ml, about 1 to about 3 mg/ml, about 1.2 to about2.8 mg/ml, about 1.4 to about 2.6 mg/ml, or about 1.6 to 2.4 aboutmg/ml.
 21. The suspension of claim 15, wherein extracellular hemoglobinlevels do not exceed 0.2 g/dl for at least 72 hours when the suspensionis maintained between 2 and 8° C.
 22. The suspension of claim 15,wherein less than 1% hemolysis occurs in 72 hours when the suspension ismaintained between 2 and 8° C.
 23. The suspension of claim 15, having anosmolarity of between about 270 and about 350 mOsm/l.
 24. The suspensionof claim 15, wherein the erythrocytes are human erythrocytes or aregenerated from human stem cells capable of becoming erythrocytes. 25.The suspension of claim 24, wherein the stem cells have been transformedto express the magnesium-independent ADI.
 26. The suspension of claim15, wherein the reduction in blood arginine levels provides a clinicalbenefit in the treatment and/or prevention of any one or all of thefollowing: arginase-1 deficiency (A1D), arginine-dependent cancers,septic shock, and angiogenesis-related diseases.
 27. The suspension ofclaim 15, packaged as a single dose in a container suitable for bloodtransfusion, said dose having a volume of between about 150 ml to about350 ml.
 28. The suspension of claim 27, wherein the dose comprisesbetween about 50 to about 3500 units (U) of ADI enzymatic activity perkilogram of patient body weight.
 29. The suspension of claim 15,prepared according to a method comprising the following steps: 1)suspending erythrocytes in an isotonic solution; 2) exposing thesuspension to a cooled hypotonic solution between +1 and +8° C., whereinthe hypotonic solution and the suspension are separated by a dialysismembrane; 3) encapsulating the ADI at a temperature between +1 and +8°C.; and 4) resealing the erythrocytes in an isotonic or hypertonicsolution at a temperature between about +30 and +42° C.
 30. Thesuspension of claim 15, prepared according to a method comprising thefollowing steps: 1) suspending a pellet of erythrocytes in an isotonicsolution at a hematocrit level equal to or greater than 65%, between +1and +8° C.; 2) lysing the erythrocytes at a temperature between +1 and+8° C. by passing the suspension into a dialysis device and exposing theerythrocytes to hypotonic conditions; 3) encapsulating the erythrocytesby adding the ADI to the suspension before and/or during the lysingstep, at a temperature between +1 and +8° C.; and 4) resealing theerythrocytes under isotonic or hypertonic conditions at a temperaturebetween +30 and +42° C.
 31. The suspension of claim 15, preparedaccording to a method comprising the following steps: 1) encapsulatingthe ADI inside the erythrocytes by placing the erythrocytes into contactwith a hypotonic medium, contacting the erythrocytes with the ADI, andresealing the erythrocytes; 2) preparing a suspension comprising theresealed erythrocytes of step 1 having an osmolality of between about290 and about 330 mOsmol/kg; 3) incubating the suspension of step 2 atan osmolality of between about 290 and about 330 mOsmol/kg, for a periodgreater than or equal to 30 minutes; 4) removing the liquid medium ofthe incubated suspension of step 3; 5) suspending the erythrocytesobtained under step 4 into a solution suitable for administration of thesuspension to a patient.
 32. The suspension of claim 15, wherein thesuspension is a stabilized erythrocyte suspension.
 33. The suspension ofclaim 15, wherein the suspension is a ready-to-use stabilizederythrocyte suspension.
 34. The suspension of claim 15, wherein the ADIis co-factor independent.